Antiinflammatory Activity of Syzygium caryophyllatum and Syzygium nervosum (Myrtaceae)

In the present study examined, the anti-inflammatory potential of aqueous, hexane, ethyl acetate and ethanolic leaf extracts of Syzygium caryophyllatum and S yzygium nervosum (Family: Myrtaceae) was carried out according to method of Mizushima et al ( 1968) and Sakat et al (2010) with slight modifications. The absorbance was checked at 660nm against the blank. Acetyl Salicylic acid was used as positive control and water as negative control. The experiment was performed in triplicates for all the test samples. Percentage of inhibition was calculated. Our results indicate that the leaf extracts of Syzygium caryophyllatum and Syzygium nervosum were showed potent anti-inflammatory activity than the standard Acetyl Salicylic acid.


Introduction
Inflammation and pain are clinical conditions present in most pathologies, being among main sources of dysfunctional and disabling conditions, requiring pharmacological intervention (Nathan and Ding, 2010;Henschke et al., 2015).Currently the most commonly used medications are steroidal and non steroidal anti-inflammatory drugs (NSAIDs), and central-acting analgesics (Conforti et al., 2009;Kunanusorn et al., 2009).In fact, NSAIDs are among most widely used medications due to their efficacy for a wide range of pain and inflammatory conditions.However, their long-term administration may induce several adverse effects such gastro-intestinal ulcers, hepatotoxicity, bleeding, renal disorders, and immunosuppression (Henzen, 2003;Wirtha et al., 2006).Opiates are most effective in cases of moderate to severe pain, although requires the clinical management of risks associated with side effects, abuse and dependence (Rosenblum et al., 2008).Therefore, development of more powerful and safe anti-inflammatory and analgesic drugs is still needed as alternatives to these drug limitations (Dharmasiri et al., 2003;Kumara, 2001).Plants inserted in traditional medicine have interested scientific community as a source of new bioactive substances discovery for human disorders treatment (Novais et al., 2003).In this sense, plants with therapeutic potential are a promising strategy for the development of anti-inflammatory drugs in search of a better therapy, reinforcing the importance of ethnopharmacological knowledge (Gupta et al., 2006;Hegde et al., 2014) The main cause of inflammation is denaturation of protein.Anti-inflammatory drugs like phenybutazone have been found to posses' ability to thermally induce protein denaturation (Mizushima and Kobayashi, 1968).The ability of the test compound to inhibit protein denaturation was studied as a part of study on the mechanism of the anti-inflammatory activity.Inflammation is the host response to trauma or as the defense mechanism against invasive organisms which eventually lead to redness, pain, swelling and temperature that evokes inflammatory cells (macrophages, neutrophils, monocytes, dendritic and mast cells) to invade the site of infection or wounds establishing an 'inflammatory microenvironment' that leads to the death and degradation on the organism, agent or affected cells and eventual restoration of cellular or organ repair process (Mitchell, 2016).Plants containing polysaccharides are the most potent in curing inflammatory diseases (Chandrika et al., 2016).

Materials and method: Inhibition of protein denaturation:
Inhibition of protein denaturation of extracts was carried out according to method of Mizushima et al (1968) and Sakat et al (2010) with slight modification.100µL of test sample was added with 500µL of 1% BSA.The mixture was incubated for 10 minutes at 37ºC.All the tubes containing reaction mixture were incubated in water bath at 51ºC for 20 minutes.At the end of the incubation the tubes were cooled under room temperature and the absorbance was checked at 660nm against the blank.Acetyl Salicylic acid was used as positive control and water as negative control.The experiment was performed in triplicates for all the test samples.Percentage of inhibition was calculated by means of the formula

Results
The denaturation of biological proteins causes inflammation.The denaturation pathways can be by acidic (or) alkaline reactions, heat treatment, radiation reactions, etc.Proteins lose their complex tertiary structure because of the externally induced stress under the above mentioned conditions thus leading to denaturation.The in vitro anti inflammatory capability of S.caryophyllatum and S.nervosum leaf extracts (Aqueous, ethanol, ethyl acetate and hexane) were initially subjected to protein denaturation method using different concentration (20 µg/mL -100 µg/mL ) for extracts as well as standard (Acetyl Salicylic acid) .The data evident that all the extracts (Aqueous, ethanol, ethyl acetate and hexane) of both the species were revealed prominent anti-inflammatory profile (Table 1 and 2).

In vitro anti-inflammatory activity of S.caryophyllatum leaf extracts
The anti-inflammatory activity, ability of extract to inhibit protein denaturation of aqueous extract of S.caryophyllatum exhibit good inhibitory activity against protein denaturation with maximum inhibition 99.37 % at 100 µg/mL concentration followed by 95.33 % of inhibition at 80 µg/mL concentration and minimum inhibition percentage 84.66%was found at 20 µg/mL concentration.
In ethanol extract, the maximum inhibition 98.96% was observed at 100 µg/mL concentration followed by 94.56% of inhibition was found in 80 µg/mL concentration and minimum percentage 82.1% was observed at 20 µg/mL concentration.
The ethyl acetate extract showed maximum percentage of inhibition 93.98% at 100 µg/mL concentrations followed by 90.67 % at 80 µg/mL concentrations and minimum percentage 80.3% was observed at 20 µg/mL concentrations.
In hexane extract maximum percentage of inhibition was observed 91.28% at 100 µg/mL concentrations followed by 87.37 % at 80 µg/mL concentrations and minimum percentage 77.14% was observed at 20 µg/mL concentrations.

In vitro anti-inflammatory activity of S.nervosum leaf extracts
In S. nervosum, the results showed (Table .2 & Fig. 1-4) that the antiinflammatory activity, ability of extracts (Aqueous, ethanol, ethyl acetate and hexane) to inhibit protein denaturation varied to a great extent at various concentrations (20µg/mL -100 µg/mL).
In S. nervosum leaf extracts the inhibition percentage is ranged from 77.29 % to 98.34%.The maximum percentage of inhibition was found at 100 µg/mL concentration of ethanol extract 98.34% followed by 94.39% of inhibition at 100 µg/mL concentration of aqueous extract and minimum was found at 20 µg/mL concentration of aqueous extract 77.29%.
In aqueous leaf extracts the maximum inhibition 94.39% was observed at 100 µg/mL concentration followed by 89.12% of inhibition in 80 µg/mL concentration and minimum percentage 77.29%was observed at 20 µg/mL concentration.
In ethanol extract, the maximum inhibition 98.34% was observed at 100 µg/mL concentration followed by 93.78% of inhibition in 80 µg/mL concentration and 78.19% was observed at 20 µg/mL concentration.The ethyl acetate extract showed maximum percentage of inhibition 92.73% at 100 µg/mL concentrations followed by 88.73 % at 80 µg/mL concentrations and minimum percentage 79.39% at 20 µg/mL concentrations.
In hexane extract maximum percentage of inhibition was observed 93.77% at 100 µg/mL concentrations followed by 89.32 % at 80 µg/mL concentrations and minimum percentage (78.49%) was observed at 20 µg/mL concentrations.
Among all the leaf extracts of S.caryophyllatum and S.nervosum, the maximum percentage of inhibition was found in ethanol extract of S. caryophyllatum followed by ethanol extract of S.nervosum and minimum was observed in hexane extracts of S. caryophyllatum.These results indicate that the leaf extracts of S.caryophyllatum and S.nervosum were showed potent anti-inflammatory activity.

Discussion
Inflammation is immensely complex and captivating.It is an elementary way in which the living tissue retaliates to injury, infection or irritation by engendering the cardinal signs i.e., calor, dolor, function laesa, rubor and tumor (Tracey, 2002).Basically inflammation is categorized into two formsacute (rapid stimuli to injury by furnishing plasma proteins and leukocytes to the active site of injury) and chronic inflammation (perpetuated period of inflammation) (Kim, 2007 andRommel, 2007).Generally, acute inflammation leads to appendicitis, bronchitis, dermatitis, meningitis, sinusitis, sore throat and tonsillitis, while chronic inflammation causes aging, Alzheimer's, asthma, atherosclerosis, cancer, Crohn's disease, hepatitis, peptic ulcer, periodontitis, psoriasis, rheumatoid arthritis, sclerosis, sepsis and tuberculosis diseases (Libby, 2002).On the other hand, naturally occurring anti inflammatory extracts or metabolites are preferred due to lesser side effects.
In the present study, preliminary screening for anti inflammatory activity was performed with protein denaturation method using bovine serum albumin protein.The tested extracts produced effective inhibitory profile towards protein denaturation.Among all the tested extracts, the S.caryophyllatum and S.nervosum depicted prominent in vitro anti inflammatory activity, which were higher than the standard (Acetyl Salicylic acid).

Conclusion
In vitro anti-inflammatory was studied by inhibition of protein denaturation (BSA).Our results were showed the higher proteinase inhibitory action of leaf extracts of S.caryophyllatum (46.62 ± 0.793mg GAE/dw) and S.nervosum (59.96 ± 0.656 mg GAE/dw) (Table 1) was higher than the A. marmelos and O. sanctum were observed as 74.45 µg/mL and 49.70 µg/mL , respectively (Reshma et al., 2014).These results indicate that the leaf extracts of S.caryophyllatum and S.nervosum were showed potent antiinflammatory activity than the standard Acetyl Salicylic acid.